Tetrahydrobiopterin is a required cofactor for the three aromatic amino acid hydroxylases that hydroxylate phenylalanine, tyrosine and tryptophan. Tissue levels of tetrahydrobiopterin are regulated mainly by the activity of the first enzyme on the de novo biosynthetic pathway, GTP cyclohydrolase I (GTPCH). Tetrahydrobiopterin is also required by the three isoforms of nitric oxide synthase, inducible, constitutive, and endothelial, and limits production of nitric oxide, endothelial derived relaxing factor, by macrophages, neurons, and endothelial cells, respectively. Availability of intracellular tetrahydrobiopterin likely plays a role in controlling nitric oxide-dependent vascular tone under normal and pathological conditions. Sphingolipid metabolites, ceramide, sphingosine, and sphingosine-1-phosphate, play important signaling roles in many biological processes, including cell growth, death, and differentiation. We previously showed that although ceramide plays a role in TNF-alpha induction of nitric oxide synthase and nitric oxide production in glial cells, TNF-alpha stimulates GTP cyclohydrolase activity and tetrahydrobiopterin synthesis by a ceramide independent mechanism. We have now found that activation of sphingosine kinase and generation of intracellular sphingosine-1-phosphate, a metabolite of ceramide, regulates GTPCH expression and tetrahydrobiopterin synthesis. This is the first indication that the pathways of NO and tetrahydrobiopterin synthesis may be regulated via divergent signaling pathways involving different sphingolipid metabolites. Furthermore, overexpression of sphingosine kinase protects neuronal cells from apoptosis induced by trophic factor withdrawal by inhibiting activation of caspases and of the MAP kinases, JNK and p38. In a continuing and highly successful collaboration on sphingolipid signaling with Spiegel?s lab at Georgetown University, a major breakthrough in understanding of sphingosine-1-phosphate signaling, which was published in Science in March 2001, was our discovery that a growth factor could transactivate a sphingosine-1-phosphate cell surface receptor by stimulation of sphingosine kinase and that this was a critical event regulating cell motility and also has important implications for blood vessel formation. In other related studies, we showed that: EDG-8, which is highly expressed in the nervous system, is a receptor for sphingosine-1-phosphate; sphingosine kinase is present in the nucleus of cells where it may play a signaling role; and sphingosine, the precursor of sphingosine-1-phosphate, is involved in apoptosis induced by chemotherapeutic drugs.